Glycosylation is important for the bioactivity of many natural products, including antibacterial compounds such as the polyketide erythromycin A and the glycopeptide vancomycin, and antitumour compounds such as the aromatic polyketide daunorubicin and the glycopeptide-polyketide bleomycin. Polyketides are a large and structurally diverse class of natural products that includes many compounds possessing antibiotic or other pharmacological properties, such as erythromycin, tetracyclines, rapamycin, avermectin, monensin, epothilones and FK506. In particular, polyketides are abundantly produced by Streptomyces and related actinomycete bacteria. They are synthesised by the repeated stepwise condensation of acylthioesters in a manner analogous to that of fatty acid biosynthesis. The greater structural diversity found among natural polyketides arises from the selection of (usually) acetate or propionate as “starter” or “extender” units; and from the differing degree of processing of the β-keto group observed after each condensation. Examples of processing steps include reduction to β-hydroxyacyl-, reduction followed by dehydration to 2-enoyl-, and complete reduction to the saturated acylthioester. The stereochemical outcome of these processing steps is also specified for each cycle of chain extension. The polyketide chains are usually cyclised in specific ways and subject to further enzyme-catalysed modifications to produce the final polyketide. Naturally-occurring peptides produced by non-ribosomal peptide synthetases are likewise synthesised by repeated stepwise assembly, in this case of activated amino acids, and the chains produced are similarly subject to further modifications to produce the fully bioactive molecules. Mixed polyketide-peptide compounds, hereinafter defined as incorporating both ketide and amino acid units, are also known and their bioactivity is also influenced by their pattern of glycosylation and other modification. The compounds so produced are particularly valuable because they include large numbers of compounds of known utility, for example as anthelminthics, insecticides, immunosuppressants, antifungal or antibacterial agents.
Streptomyces and closely-related genera of filamentous bacteria are abundant producers of polyketide metabolites. Although large numbers of therapeutically important polyketides have been identified, there remains a need to obtain novel polyketides that have enhanced properties or that possess completely novel bioactivity. The inexorable rise in the incidence of pathogenic organisms with resistance to antibiotics such as 14-membered macrolides or glycopeptides represents a significant threat to human and animal health. Current methods of obtaining novel polyketide metabolites include large-scale screening of naturally-occurring strains of Streptomyces and other organisms, either for direct production of useful molecules, or for the presence of enzymatic activities that can bioconvert an existing polyketide, which is added to the growth medium, into specific derivatives. These procedures are time-consuming and costly, and biotransformation using whole cells may in addition be limited by side-reactions or by a low concentration or activity of the intracellular enzyme responsible for the bioconversion. Given the complexity of bioactive polyketides, they are not readily amenable to total chemical synthesis in large scale. Chemical modification of existing polyketides has been widely used, but many desirable alterations are not readily achievable by this means.
Meanwhile, methods have been developed for the biosynthesis of altered polyketides and non-ribosomally-synthesised polypeptides by the engineering of the corresponding genes encoding the polyketide synthases and polypeptide synthetases respectively. The biosynthesis of polyketides is initiated by a group of chain-forming enzymes known as polyketide synthases. Two classes of polyketide synthase (PKS) have been described in actinomycetes. One class, named Type I PKSs, represented by the PKSs for the macrolides erythromycin, oleandomycin, avermectin and rapamycin, consists of a different set or “extension module” of enzymes for each cycle of polyketide chain extension (Cortes, J. et al. Nature (1990) 348:176-178). The term “extension module” as used herein refers to the set of contiguous domains, from a β-ketoacyl-ACP synthase (“KS”) domain to the next acyl carrier protein (“ACP”) domain, which accomplishes one cycle of polyketide chain extension.
In-frame deletion of the DNA encoding part of the ketoreductase domain in module 5 of the erythromycin-producing PKS (also known as 6-deoxyerythronolide B synthase, DEBS) has been shown to lead to the formation of erythromycin analogues 5,6-dideoxy-3-α-mycarosyl-5-oxo-erythronolide B, 5,6-dideoxy-5-oxoerythronolide B and 5,6-dideoxy, 6 β-epoxy-5-oxoerythronolide B (Donadio, S. et al. Science (1991) 252:675-679). Likewise, alteration of active site residues in the enoylreductase domain of module 4 in DEBS, by genetic engineering of the corresponding PKS-encoding DNA and its introduction into Saccharopolyspora erythraea, led to the production of 6,7-anhydroerythromycin C (Donadio, S. et al. Proc Natl. Acad. Sci. USA (1993) 90:7119-7123). WO 93/13663 describes additional types of genetic manipulation of the DEBS genes that are capable of producing altered polyketides. However many such attempts are reported to have been unproductive (Hutchinson, C. R. and Fujii, I. Annu. Rev. Microbiol. (1995) 49:201-238, at p. 231).
WO 98/01546 describes the engineering of hybrid Type I PKS genes which utilise portions of PKS genes derived from more than one natural PKS, particularly derived from different organisms, and the use of such recombinant genes for the production of altered polyketide metabolites.
The second class of PKS, named Type II PKSs, is represented by the synthases for aromatic compounds. Type II PKSs contain only a single set of enzymatic activities for chain extension and these are re-used in successive cycles (Bibb, M. J. et al. EMBO J. (1989) 8:2727-2736; Sherman, D. H. et al. EMBO J. (1989) 8:2717-2725; Femandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278-19290). The “extender” units for the Type II pKSs are usually acetate units, and the presence of specific cyclases dictates the preferred pathway for cyclisation of the completed chain into an aromatic product (Hutchinson, C. R. and Fujii, I. Annu. Rev. Microbiol. (1995) 49:201-238). Hybrid polyketides have been obtained by the introduction of clones Type II PKS gene-containing DNA into another strain containing a different Type II PKS gene cluster, for example by introduction of DNA derived from the gene cluster for actinorhodin, a blue-pigmented polyketide from Streptomyces coelicolor, into an anthraquinone polyketide-producing strain of Streptomyces galileus (Bartel, P. L. et al. J. Bacteriol. (1990) 172:4816-4826).
The minimal number of domains required for polyketide chain extension on a Type II PKS when expressed in a Streptomyces coelicolor host cell (the “minimal PKS”) has been defined for example in WO 95/08548 as containing the following three polypeptides which are products of the act I genes: first KS; secondly a polypeptide termed the CLF with end-to-end amino acid sequence similarity to the KS but in which the essential active site residue of the KS, namely a cysteine residue, is substituted either by a glutamine residue, or in the case of the PKS for a spore pigment such as the whiE gene product (Chater, K. F. and Davis, N. K. Mol. Microbiol. (1990) 4:1679-1691) by a glutamic acid residue; and finally an ACP. The CLF has been stated for example in WO 95/08548 to be a factor that determines the chain length of the polyketide chain that is produced by the minimal PKS. However, it has been found (Shen, B. et al. J. Am. Chem. Soc. (1995) 117:6811-6821) that when the CLF for the octaketide actinorhodin is used to replace the CLF for the decaketide tetracenomycin in host cells of Streptomyces glaucescens, the polyketide product is not found to be altered from a decaketide to an octaketide. An alternative nomenclature has been proposed in which KS is designated KSα and CLF is designated KSβ, to reflect this lack of confidence in the correct assignment of the function of CLF (Meurer, G. et al. Chemistry and Biology (1997) 4:433-443). International Patent Application WO 00/00618 has recently shown that CLF and its counterpart in Type I PKS multienzymes, the so-called KSQ domain, are involved in initiation of polyketide chain synthesis. WO 95/08548 for example describes the replacement of actinorhodin PKS genes by heterologous DNA from other Type II PKS gene clusters, to obtain hybrid polyketides.
This ability to engineer PKS genes of both Type I and Type II raises the possibility of combinatorial biosynthesis of polyketides to produce diverse libraries of novel natural products which may be screened for desirable bioactivities. However, the aglycones produced by the recombinant PKS genes may be only partially, or not at all, processed by glycosyltransferases and other modifying enzymes into analogues of the mature polyketides. There is therefore an additional need to provide processes for efficient conversion of such novel aglycones into specific glycosylated products. Further, the invention of efficient processes for glycosylation would provide a new means to increase very significantly the diversity of combinatorial polyketide libraries, by utilisation of recombinant cells containing alternative cloned glycosyltransferases and alternative complements of activated sugars.
The well-known influence of glycosylation on biological activity has encouraged intensive research into the genes and enzymes governing the synthesis and attachment of specific sugar units to polyketide and polypeptide metabolites (for a review see Trefzer, A. et al. Natural Products Reports (1999) 16:283-299). Surveys of such metabolites have revealed a high diversity in the type of glycosyl substitution that is found, including a very large number of different deoxyhexoses and deoxyaminohexoses (see for a review Liu, H.-W. and Thorson, J. S. Annu. Rev. Microbiol. (1994) 48:223-256) review). The sequencing of biosynthetic gene clusters for numerous glycosylated polyketides and peptides has revealed the presence of such sugar biosynthetic genes, and also genes encoding the glycosyltransferases that transfer the glycosyl group from an activated form of the sugar, eg dTDP- or dUDP-forms, to the aglycone acceptor. For example, the eryB genes and the eryC genes of the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea have been identified as involved in the biosynthesis and attachment of respectively, L-mycarose and D-desosamine to the aglycone precursor of erythromycin A (Dhillon, N. et al., Mol. Microbiol. (1989) 3:1405-1414; Haydock et al. Mol. Gen. Genet. (1991) 230:120-128; Salah-Bey, K. et al. Mol Gen. Genet (1998) 257:542-553; Gaisser, S. et al., Mol. Gen. Genet. (1998) 258:78-88; Gaisser, S. et al. (1997) Mol. Gen. Genet. 256: 239-251; Summers, D. et al. Microbiology (1997) 143: 3251-3262). Both WO 97/23630 and WO 99/05283 describe the preparation of an altered erythromycin by deletion of a specific sugar biosynthetic gene, so that an altered sugar becomes attached to the aglycone. Thus WO 99/05283 describes low but detectable levels of erythromycins in which for example desosamine is replaced by mycaminose (eryCIV knockout), or desmethylmycarosyl erythromycins (eryBIII knockout) are produced.
Meanwhile methymycin analogues have been produced in which desosamine has been replaced by D-quinuvose (Borisova, S. A. et al. Org. Lett. (1999) 1:133-136), or through the incorporation of the calH gene of the calicheamycin gene cluster from Micromonospora echinospora into the methymycin producing strain (Zhao, L. et al. J. Amer. Chem. Soc. (1999) 121:9881-9882). Similarly, hybrid glycopeptides have been produced by using cloned glycosyltransferases from the vancomycin-producing Amycolatopsis orientalis to add D-xylose or D-glucose to aglycones of closely-related glycopeptides according to U.S. Pat. No. 5,871,983 (1999) (Solenberg, P. et al. Chem. Biol (1997) 4:195-202). Hybrid aromatic polyketides have also been produced, by interspecies complementation of a mutant individual sugar biosynthetic gene-with a similar gene with a different stereospecificity. Thus instead of the natural daunosamine, 4′epi-daunosamine is produced in recombinant Streptomyces peucetius and attached by the daunosamine glycosyltransferase to the aglycone to yield the antitumour derivative epirubicin in place of doxorubicin (Madduri, K. et al. Nature Biotechnology (1998) 16:69-74). In all these cases, the specificity of the glycosyltransferase allowed the substitution of an alternative activated sugar, but the aglycone and glycosyltransferase were not heterologous to each other. It has been found that when oleandrose glycosyltransferase oleG2 of Streptomyces antibioticus is cloned into the erythromycin-producing Saccharopolyspora erythraea, in addition to other products, the novel erythromycin in which cladinose/mycarose at C-3 is replaced by rhamnose, was obtained (Doumith, M. et al, Mol. Microbiol. (1999) 34:1039-1048). It was assumed that the activated rhamnose is produced by the host cells, and is recruited by the oleG2 glycosyltransferase in competition with the activated mycarose known to be present.